High-Coverage Strategy for Multi-Subcellular Metabolome Analysis Using Dansyl-Labeling-Based LC-MS/MS

Abstract

Subcellular compartmentalization ensures orderly and efficient intracellular metabolic activities in eukaryotic life. Investigation of the subcellular metabolome could provide in-depth insight into cellular biological activities. However, the sensitive measurement of multi-subcellular metabolic profiles is still a significant challenge. Herein, we present a comprehensive subcellular fractionation, characterization, and metabolome analysis strategy. First, six subcellular fractions including nuclei, mitochondria, lysosomes, peroxisomes, microsomes, and cytoplasm were generated from a single aliquot of liver homogenate. Then, a dansyl-labeling-assisted liquid chromatography−tandem mass spectrometry (LC-MS/MS) method for measuring 151 amino/ phenol-or carboxyl-containing metabolites in the subcellular fractions was established and validated. Last, the strategy was applied to a rat model of carbon tetrachloride (CCl 4 )-induced acute liver injury (ALI). The metabolic profile of individual organelles was compared with that of the liver. Interestingly, many unique changes were observed specifically in organelles, while the liver failed to capture these changes. This result indicates that metabolic investigation at the tissue level might lead to erroneous results due to the leveling effect. Our study demonstrates a feasible approach for the broad-spectrum-targeted metabolic profiling of multi-subcellular fractions, which can be of great use in driving our further understanding of intracellular metabolic activities in various physical and pathological conditions.

An optimal combination of four active components in Huangqin decoction for the synergistic sensitization of irinotecan against colorectal cancer

“…To investigate the underlying mechanism of the synergistic anticancer effect of Cmb8, we employed a targeted metabolomics method [ 11 ] covering 151 metabolites to study metabolic differences among the Model, Cmb8, CPT-11, and Cmb8 + CPT-11 groups. A total of 44 differential metabolites were obtained by comparing each group with the Model group using the criteria of fold change > 1.3 and P < 0.05 (Fig.…”

“…In this study, a targeted metabolomics method covering 151 metabolites, as recently published by our group [ 11 ], was employed to investigate metabolic changes following drug treatment. Frozen tumor samples (~ 30 mg) were placed into pre-cooled homogenization tubes, to which extraction solvent mixture ethanol-acetonitrile (1:1, v/v) was added at a ratio of 30 μL per milligram of tissue.…”

“…Metabolomic analysis was conducted using a Shimadzu Nexera UPLC system interfaced with an 8060 triple quadruple mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization source. Details for instrument conditions, column, mobile phase, and MS parameters are provided in the recent publication [ 11 ] as well as in the Extended methods section in the Supplementary Material .…”